calcimycin and Lung-Neoplasms

Pancreatic ductal and lung adenocarcinomas are the most common and prevalent types of human neoplasms with a greater than 80% mortality rate. The poor prognosis of both these cancers are likely due to the absence of valid approaches for early detection, the frequency of its metastases at the time of diagnosis, frequent recurrence after surgery, and poor responsiveness to chemotherapy. Most notably, the early development of pancreatic intraepithelial neoplasia and lung lesions is suggested to be the result of a mutation in the K-ras (G12D) oncogene. Tumor necrosis factor-related-apoptosis-inducing-ligand (TRAIL) has been shown to have great potential for the treatment of most human tumor cells, while leaving normal cells unharmed. However, some cancers show resistance to TRAIL treatment, leaving a gap in the understanding of its exact etiology.. TRAIL-induced resistance to cell death was investigated in pancreatic and lung cancer cell lines. Cell survival was determined by SRB and apoptosis by ELISA-based cell death assay. Activation of bid and caspases were evaluated by Western blotting.. Our study demonstrated that TRAIL significantly suppressed cell survival, by inducing apoptosis in a dose-dependent manner, in the pancreatic cancer BxPC-3 (wild type G12) and lung cancer A549 (G12S) cell lines. In contrast, Panc-1 pancreatic and SK-LU-1 lung cancer cell lines, which have a mutated (G12D) K-ras genotype, were resistant to the actions of TRAIL.. This study demonstrates an association between TRAIL resistance to apoptosis in human pancreatic and lung cancer cell lines and G12D K-ras(12) mutation.

Topics: Amino Acid Substitution; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Calcimycin; Caspases; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Genes, ras; Humans; Lung Neoplasms; Pancreatic Neoplasms; Point Mutation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca(2+), stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.

Topics: Animals; Calcimycin; Calcium; Cardiac Myosins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Down-Regulation; Gene Expression; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 4 or More Rings; Homeodomain Proteins; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Myosin Light Chains; Neoplasm Invasiveness; Phosphodiesterase 5 Inhibitors; Phosphorylation; POU Domain Factors; Promoter Regions, Genetic; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; RNA, Small Interfering; Transplantation, Heterologous

Glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, plays a critical role in chemotherapy resistance in a variety of cancers. In this study, we investigated the up-regulation of GRP78 induced by A23187 and its association with the chemotherapeutical sensibility to cisplatin in human lung cancer cell line SPCA1.. SPCA1 cells were pretreated with A23187 at different concentrations. The expression of GRP78 at the mRNA level was analyzed by RT-PCR; the expression of GRP78 at the protein level was determined by Western blotting and immunofluorescence assay. Cell survival was determined by MTT assay. Cell apoptosis was analyzed by flow cytometry.. The expression of GRP78 at both the mRNA and protein levels was obviously induced by A23187 in SPCA1 cells, with an elevation of GRP78 by 2.1-fold at the mRNA level and by 3.8-fold at the protein level compared to the control. There was a dose-dependent response. Survival curve analysis demonstrated that A23187 induction caused a significant reduction of survival for the cells subjected to cisplatin treatment (P < 0.05). After treatment by cisplatin, the percentage of apoptotic cells in the A23187 pretreated group increased about three fold compared with the control group ((27.53 ± 4.32)% vs. (9.25 ± 3.64)%, P < 0.05).. A23187 treatment was fairly effective for the induction of GRP78 in SPCA1 cells at both the mRNA and protein levels. To a certain extent, GRP78 up-regulation by A23187 was associated with the enhancement of drug sensitivity to cisplatin in human lung cancer cell line SPCA1.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Calcimycin; Cell Line, Tumor; Cisplatin; Endoplasmic Reticulum Chaperone BiP; Flow Cytometry; Heat-Shock Proteins; Humans; Lung Neoplasms; Reverse Transcriptase Polymerase Chain Reaction

The major challenge of chemotherapy is the disease resistance for many lung cancer patients. Integrated microfluidic systems offer many desirable characteristics and can be used in cellular biological analysis. This work aimed to study the correlation between the expression of Glucose Regulated Protein-78 (GRP78) and the resistance to anticancer drug VP-16 in human lung squamous carcinoma cell line SK-MES-1 using an integrated microfluidic gradient chip device. We used A23187, a GRP78 inducer, with a gradient concentration in the upstream network of the device to induce the expression of GRP78 in the cells cultured in the downstream before the addition of VP-16. The expression of GRP78 was detected by immunofluorescence, the apoptosis for the cells treated by VP-16 was assessed morphologically by 4',6-diamidino-2-phenylindole (DAPI) staining. The results indicated that the expressions of GRP78 increased greatly for the cells under the induction of A23187 with a dose-depended manner, while the percentage of apoptotic cells decreased significantly after being treated by VP-16. Our results from this study confirmed the role of GRP78 played in the chemotherapy resistance to VP-16 in SK-MES-1 cell line, suggesting that the integrated microfluidic systems may be an unique approach for characterizing the cellular responses.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Calcimycin; Cell Line, Tumor; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Etoposide; Fluorescent Antibody Technique; Fluorescent Dyes; Heat-Shock Proteins; Humans; Image Processing, Computer-Assisted; Indoles; Ionophores; Lung Neoplasms; Microfluidic Analytical Techniques; Reproducibility of Results

The upregulation of GRP78 and GRP94 under the induction of A23187 and its function in drug resistance to etoposide (VP-16) was investigated in human lung cancer cell line SK-MES-1. The expression of GRP78 and GRP94 induced by A23187 at different concentrations was analyzed by RT-PCR and Western blotting. Cell survival to VP-16 was determined using a colony-formation assay. The expression of GRP78 and GRP94 in the cells was found to correspond well with the cell survival to VP-16. The results showed that upregulation of GRP78 and GRP94 can significantly confer the chemoresistance to VP-16 in human lung cancer cell line SK-MES-1.

Topics: Antineoplastic Agents, Phytogenic; Blotting, Western; Calcimycin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Etoposide; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Lung Neoplasms; Membrane Glycoproteins; Molecular Chaperones; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Stem Cell Assay; Up-Regulation

Chemotherapy resistance remains a major obstacle for the treatment of small cell lung cancer (SCLC). Glucose-regulated protein 78 (GRP78), an endoplasmic reticulum chaperone, plays a critical role in chemotherapy resistance in some cancers. However, whether the suppression of the chaperone can enhance the sensitivity of chemotherapy in SCLC is still unclear.. The SCLC NCI-H446 cells were divided into three groups: BAPTA-AM-->A23187-treated group, A23187-treated group and control-group. Immunofluorescence, western blot and RT-PCR were used to assess the expression of GRP78 at both protein and mRNA levels. Cell apoptosis and the cell cycle distributions of the cells were analyzed by flow cytometry in order to evaluate the therapeutic sensitivity to VP-16.. The expression of GRP78 at both protein and mRNA levels in the BAPTA-AM-->A23187-treated cells dramatically decreased as compared to that in both A23187-treated and control groups. After treatment by VP-16, the percentage of apoptotic cells in BAPTA-AM-->A23187-treated cells were: 33.4 +/- 1.01%, 48.2 +/- 1.77%, 53.0 +/- 1.43%, 56.5 +/- 2.13%, respectively, corresponding to the concentrations of BAPTA-AM 10, 15, 25, 40 microM, which was statistically significant high in comparison with the A23187-treated group and untreated-group (7.18 +/- 1.03% and 27.8 +/- 1.45%, respectively, p < 0.05). The results from analysis of cell cycle distribution showed that there was a significantly decreased in G1 phase and a dramatically increased in S phase for the BAPTA-AM-->A23187-treated cells as compared with the untreated cells.. BAPTA-AM is a strong inhibitor of GRP78 in the NCI-H446 cell line, the down-regulation of GRP78 can significantly increase the sensitivity to VP-16. The suppression of GRP78 may offer a new surrogated therapeutic approach to the clinical management of lung cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Calcimycin; Cell Cycle; Cell Line, Tumor; Down-Regulation; Egtazic Acid; Endoplasmic Reticulum Chaperone BiP; Etoposide; Flow Cytometry; Fluorescent Antibody Technique; Heat-Shock Proteins; Humans; Lung Neoplasms; Molecular Chaperones; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Small Cell Lung Carcinoma

Microchip-based systems have many desirable characteristics and can be used in much cellular biochemical analysis. Glucose-regulated protein 78 (GRP78), an endoplasmic reticulum chaperone, has a critical role in chemotherapy resistance of some cancers. This work aimed at analyzing the correlation between the expression of GRP78 and an anticancer drug, topoisomerase II inhibitor-VP-16, in human lung cancer cell line NCI-H460 using this microchip-based system. The cells were cultured on a PDMS chip, the expression of GRP78 at both protein and mRNA levels for the cells under the condition with or without the induction of A23187 were assayed by immunofluorescence and chip electrophoresis, respectively. Then the cells were treated by VP-16, percentages of apoptosis and the cycle distributions of the cells were detected by flow cytometry. The cells cultured on the PDMS attached and spread well to micro-channels with high viability. Compared with the non-induced cells, the expression of GRP78 at both protein and mRNA levels for the A23187-induced cells were increased greatly. After treatment by VP-16, the percentage of apoptotic cells decreased nearly threefold for the A23187-induced cells in contrast to the non-induced cells (13.15 +/- 3.84% versus 34.03 +/- 11.45%), and the cells distributed in S phase reduced dramatically (11.96 +/- 1.27% versus 20.76 +/- 3.05%) whereas in G(1) phase increased greatly (74.16 +/- 0.95% versus 57.06 +/- 4%). GRP78 is correlated to the resistance to VP-16 in human lung cancer cell line. The microchip-based system has the potential application and feasibility for cell culture and its functional research.

Topics: Antineoplastic Agents; Calcimycin; Capillary Electrochromatography; Cell Culture Techniques; Cell Line, Tumor; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Equipment Design; Equipment Failure Analysis; Heat-Shock Proteins; Humans; Immunoassay; Lab-On-A-Chip Devices; Lung Neoplasms; Molecular Chaperones

In human airways mucosal mast cells are under the control of inhibitory muscarinic receptors. The described experiments tested, whether the inhibitory potency of two muscarinic receptor agonists (oxotremorine, acetylcholine) becomes impaired in advanced chronic obstructive pulmonary disease (COPD). Isolated human bronchi obtained from 26 patients with lung cancer were separated into two groups. Group 1 patients suffered from moderate COPD (mean FEV1 56%; range 34-71%; mean pack years of cigarette smoking 50, range 20-96; one non-smoker). Group 2 patients had no or only a mild form of COPD; mean FEV1 was 82% (62-97%) and the number of pack years 22 (6-45; 3 non-smoker). The calcium ionophore A23187 induced a maximal histamine release of 4100+/-870 pmol/g/5 min in group 1 bronchi, in contrast to only 1730+/-240 pmol/g/5 min in group 2 bronchi (p<0.02). Oxotremorine (1 nmol/L) reduced the stimulated histamine release by 81+/-5% in group 2 bronchi, but did not produce a significant effect in group 1 bronchi (11+/-14%). In conclusion, the present experiments show an enhanced histamine release in advanced COPD, which can be explained by a dysfunction of inhibitory muscarinic receptors.

Topics: Acetylcholine; Aged; Bronchi; Calcimycin; Female; Forced Expiratory Volume; Histamine Release; Humans; In Vitro Techniques; Ionophores; Lung Neoplasms; Male; Middle Aged; Muscarinic Agonists; Oxotremorine; Pulmonary Disease, Chronic Obstructive; Receptors, Muscarinic; Smoking; Time Factors

Preclinical data suggest that the cyclooxygenase (COX)-2/prostaglandin (PG) E2 signaling pathway plays an essential role in conferring the malignant phenotype in non-small cell lung cancer. We hypothesized that treatment with oral celecoxib, a selective COX-2 inhibitor, would favorably alter biomarkers of lung cancer risk. This study evaluated the feasibility of COX-2 inhibition as a form of chemoprevention for lung cancer.. Heavy active smokers were enrolled into a pilot study and treated with celecoxib. Bronchoscopy with bronchoalveolar lavage was performed both before and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from nonsmoking control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation.. Treatment with celecoxib significantly reduced calcium ionophore-stimulated PGE2 production from AMs recovered from smokers. AMs recovered from smokers, but not nonsmokers, were primed to produce high levels of PGE2 and interleukin (IL-10) when stimulated with lipopolysaccharide, and SC58236 significantly abrogated the production of these factors. Moreover, both plasma and bronchoalveolar lavage fluid obtained from treated subjects significantly reduced the production of PGE2 that resulted when a lung cancer cell line, A549, was stimulated with IL-1beta or A23187.. Our findings suggest that oral celecoxib is capable of inhibiting the overproduction of PGE2, as well as modulating the production of IL-10 in the lung microenvironment in individuals at risk for lung cancer.

Topics: Aged; Bronchoalveolar Lavage; Bronchoscopy; Calcimycin; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Celecoxib; Cyclooxygenase Inhibitors; Dinoprostone; Feasibility Studies; Female; Humans; Interleukin-10; Ionophores; Lipopolysaccharides; Lung Neoplasms; Macrophages, Alveolar; Male; Middle Aged; Pilot Projects; Pyrazoles; Smoking; Sulfonamides; Tumor Cells, Cultured

A novel approach to reducing organ toxicity of anticancer agents is the application of nontoxic glucuronide prodrugs from which the active drug is released by human beta-glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in beta-glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human beta-glucuronidase expression are not available. Preliminary evidence from animal experiments points to a role of intracellular calcium in regulation of beta-glucuronidase activity. Therefore, we investigated regulation of beta-glucuronidase by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. The enzyme was characterized on activity, protein, and mRNA levels by cleavage of 4-methylumbelliferyl-beta-D-glucuronide, Western blotting, Northern blotting, and nuclear run-on transcription. Incubation of HepG2 cells with A23187 and thapsigargin, respectively, revealed a time and concentration dependent down-regulation of beta-glucuronidase activity to about 50% of the control level. This effect could also be demonstrated in several other cell lines (e.g., HL-60, ECV 304, 32M1, Caco-2/TC7). Effects on protein and mRNA levels paralleled those obtained on enzymatic activity. In line with these data, A23187 and thapsigargin decreased beta-glucuronidase transcriptional rate. Our data demonstrate regulation of human beta-glucuronidase by xenobiotics. Down-regulation of beta-glucuronidase by A23187 and thapsigargin is at least partly mediated by a transcriptional mechanism. Based on our findings, we speculate that beta-glucuronidase activity and hence bioactivation of glucuronide prodrugs in humans can be modulated by exogenous factors.

Topics: Blotting, Northern; Blotting, Western; Calcimycin; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glucuronidase; Humans; Ionophores; Lung Neoplasms; RNA, Messenger; Thapsigargin; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Calcimycin; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Communication; Cell Line; Coculture Techniques; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lung Neoplasms; Models, Biological; Neutrophils; Phospholipases A; Phospholipases A2; Tumor Cells, Cultured

Non-small cell lung cancer (NSCLC) is fatal in approximately 90% of all cases due to the failure of systemic therapy, secondary to resistance to chemotherapy. In such malignancies new therapeutic paradigms are needed. One such approach takes advantage of normal physiologic growth regulatory mechanisms, such as terminal cellular differentiation or apoptosis. Suramin, as an antineoplastic drug, has shown efficacy in the treatment of prostate cancer and is capable of promoting differentiation in several human cancer cell lines. Little is known about the differentiating effects of suramin in lung cancer. In the present investigation we evaluated the ability of suramin to induce cross-linked envelope (CLE) formation, as a common marker for squamous differentiation and apoptosis, in three representative human non-small cell lung cancer cell lines: NCI-H226 (squamous), NCI-H358 (bronchoalveolar [adenocarcinoma]), and NCI-H596 (adenosquamous). Among agents that we have tested, suramin demonstrated the unique ability to induce spontaneous CLE formation in the two cell lines with squamous features, NCI-H226 and NCI-H596. Suramin induced CLE formation was accompanied by DNA fragmentation, a marker for apoptosis, in NCI-H596 and NCI-H358, but not in NCI-H226. Stimulation of CLE formation by suramin correlated with the rapid induction of both type II transglutaminase (TG) activity and involucrin expression. These parameters were protein synthesis independent, suggesting posttranslational mechanisms of suramin activity. Induction of differentiation/apoptosis markers by suramin did not correlate with its effect on growth. Modulation of signal transduction is a likely candidate mechanism for suramin activity in lung cancer. The relationship between growth, squamous differentiation, and apoptosis is considered.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Calcimycin; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; DNA Fragmentation; Enzyme Inhibitors; Humans; Ionophores; Lung Neoplasms; Neoplasm Proteins; Protein Kinase C; Protein Precursors; Putrescine; Suramin; Transglutaminases; Tumor Cells, Cultured

We studied cellular interactions between human polymorphonuclear leukocytes (PMN) and lung cancer cell lines by investigating the influence of cancer cells on the production of leukotriene B4 (LTB4) and superoxide anion (O2-) by stimulated PMN. Of the nine cancer cell lines established from human lung cancers that we examined, H23 cells showed the highest LTA4 hydrolase activity. When PMN were stimulated by the calcium ionophore A23187 in the presence of H23 cells, the production of LTB4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), and 12(S)-hydroxyeicosatetraenoic acid (12-HETE) decreased in a dose-dependent manner. On the contrary, H23 did not inhibit O2- production by PMN. Two other cell lines (N417 and Q9) caused similar inhibition of LTB4 production by PMN. These three cancer cell lines alone did not generate any metabolites of the arachidonic acid (AA) lipoxygenase pathway or any O2- upon stimulation with A23187 alone. The addition of AA dose-dependently reversed the H23-induced inhibition of LTB4, 5-HETE, and 12-HETE production by PMN, suggesting inhibition at the phospholipase A2 (PLA2) level. Furthermore, addition of the cancer cell line Q9 inhibited 14C release from [14C]AA prelabeled PMN in a cell number-dependent manner in the buffer, with and without albumin. The supernatant of H23 cells also inhibited the production of LTB4 by PMN stimulated by A23187, as did the addition of H23 lysate or its 10(4) x g centrifugation supernatant. While neither the 10(5) x g supernatant (cytosol) nor the pellet (microsome) exhibited inhibitory activity, the combination of the separated cytosol and microsomal fractions restored the inhibitory activity. Furthermore, addition of the 10(4) x g supernatant of Q9 lysate to partially purified human cytosolic PLA2 inhibited PLA2 activity in a dose-dependent manner. Our results indicate that the lung cancer cell lines used in our study inhibit LTB4 production by human PMN through inhibition of phospholipase A2 activity, which may contribute to a predisposition to pulmonary infections in patients with lung cancer.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Calcimycin; Calcium; Coculture Techniques; Cyclooxygenase Inhibitors; Dinoprostone; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Ionophores; Leukotriene B4; Lipoxygenase; Lung Neoplasms; Neutrophils; Phospholipases A; Phospholipases A2; Subcellular Fractions; Superoxides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The mechanism of ectopic adrenocorticotrophic hormone (ACTH) secretion was examined by studies on the effects of corticotropin-releasing hormone (CRH), dexamethasone, interleukin (IL)-1 beta and 2, somatostatin, calcium ionophore A23187, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 8-bromo-cAMP on pro-opiomelanocortin (POMC) expression and ACTH secretion from a human small cell lung cancer cell line COR-L103. None of these agents except TPA and A23187 had any effect on ACTH secretion from the cell line in short (0-8 hrs) or long term (1-4 days) cultures. In long term cultures, 1-100 nM TPA stimulated ACTH secretion dose-dependently, whereas 500nM A23187 inhibited ACTH secretion completely. When the cells were incubated with 10nM TPA plus 500 nM A23187, the inhibitory action of A23187 on ACTH secretion was suppressed by TPA. These results suggest that the mechanisms of ACTH secretion by COR-L103 cells and normal pituitary cells are different.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenocorticotropic Hormone; Calcimycin; Carcinoma, Small Cell; Cell Line; Corticotropin-Releasing Hormone; Dexamethasone; Dose-Response Relationship, Drug; Humans; Immunoradiometric Assay; Interleukin-1; Interleukin-2; Kinetics; Lung Neoplasms; Pro-Opiomelanocortin; Somatostatin; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

Of 12 cell lines derived from human lung cancers, only Calu-3 cells showed high transepithelial resistance (Rte) and increases in short-circuit current (Isc) in response to mediators. Calu-3 cells formed polarized monolayers with tight junctions and Rte of approximately 100 omega.cm2. Baseline Isc was approximately 35 microA/cm2 and was increased by approximately 75 microA/cm2 on elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by isoproterenol. Flux studies showed that the increase in Isc was due to Cl- secretion. Forskolin and permeant analogues of cAMP also increased Isc. Consistent with the presence of cAMP-dependent Cl- secretion, immunoprecipitation demonstrated the presence of the cystic fibrosis transmembrane conductance regulator (CFTR). Bradykinin, methacholine, trypsin, and histamine all transiently (15-30 s) elevated Isc, probably by increasing intracellular Ca concentration. Experiments in which the basolateral membrane was permeabilized with nystatin indicated that CFTR was substantially activated under baseline conditions and that Ca-activated Cl- channels were absent from the apical membrane. We anticipate that Calu-3 cells will prove useful in the study of Cl- secretion and other functions of human airway epithelial cells.

Topics: Bradykinin; Bumetanide; Calcimycin; Cell Line; Cell Membrane; Cell Membrane Permeability; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoplasmic Granules; Desmosomes; Epithelium; Histamine; Humans; Intercellular Junctions; Isoproterenol; Kinetics; Lung Neoplasms; Membrane Proteins; Methacholine Chloride; Microscopy, Electron; Nystatin; Ouabain; Sodium; Trypsin; Tumor Cells, Cultured

Four classical and three variant small-cell carcinoma of the lung (SCCL) cell lines were examined for vasopressin and vasopressin V1a-receptor immunoreactivity. One of these classical cell lines, NCI-H345, and one variant cell line, NCI-H82, were further investigated for binding of V1 and V2 vasopressin-receptor antagonists, vasopressin-induced calcium mobilization, and vasopressin-induced thymidine uptake. All classical and variant SCCL cell lines examined contained vasopressin and vasopressin-receptors as determined by immunocytochemistry. Both NCI-H82 and NCI-H345 demonstrated similar binding patterns with the V1 and V2 vasopressin-receptor antagonists, indicating the presence of both receptor subtypes. For the classical cell line (NCI-H345), vasopressin (1 microM) induced an increase in cytosolic free calcium, while the peptide was ineffective at increasing cytosolic calcium in the variant cell line (NCI-H82). However, vasopressin (0.1 or 1 microM) was unable to stimulate thymidine uptake in the classical (NCI-H345) or variant (NCI-H82) cell lines for the conditions used. These results indicate that both classical and variant SCCL produce vasopressin, and vasopressin V1a and V2 receptors. In the variant cell line, there appears to be a disruption in the activation cascade for V1a receptors as indicated by the lack of vasopressin-induced calcium mobilization.

Topics: Calcimycin; Calcium; Carcinoma, Small Cell; Cytosol; Humans; Lung Neoplasms; Receptors, Vasopressin; Thymidine; Tumor Cells, Cultured; Vasopressins

We studied neutrophil functions in a patient with colony-stimulating activity (CSA)-producing lung cancer. A 59-year-old man had an abnormal chest X-ray and leucocytosis, predominantly with neutrophils. Pneumonectomy was performed, and the histological diagnosis of the tumour was large-cell carcinoma of the lung. The tumour induced marked granulocytosis in tumour-transplanted nude mice, and the conditioned media of the tumour contained very strong human-active CSA. Superoxide release and membrane depolarization in neutrophils stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine, were markedly enhanced in the patient. These findings suggest that CSA produced by the tumour primed neutrophil functions in vivo in the patient.

Topics: Animals; Calcimycin; Carcinoma, Non-Small-Cell Lung; Colony-Stimulating Factors; Humans; Lung Neoplasms; Male; Membrane Potentials; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neoplasm Transplantation; Neutrophils; Superoxides

In the present study, we described the effects of three different drugs, A23187 calcium ionophore (A23187), indomethacin (IND) and phorbol myristate acetate (PMA), on the secretion pattern and the metastatic potential in the low metastatic 3LL tumor cell line. The results evidence that a low molecular weight protein fraction is always secreted in an higher amount in drug treated cells than in untreated cells independently of the drug used. In addition to their effects on secretion pattern, the three drugs assayed enhance noticeably the metastatic rate in vivo of the 3LL cells. The data confirm our earlier observations showing that our high performance gel permeation chromatography (HPGPC) method is suitable for discriminating even between cells with a different degree (low and high) of metastatic potential by its chromatographic secretion pattern and that alterations in the secretion pattern from treated cells are related with changes in the degree of metastatic potential of these cells.

Topics: Animals; Calcimycin; Female; Indomethacin; Lung Neoplasms; Mice; Mice, Inbred BALB C; Molecular Weight; Neoplasm Metastasis; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Cultured Lewis Lung carcinoma cells (3LL) treated with A23187 calcium ionophore were studied by transmission electron microscopy and HPLC analysis. Results showed that A23187 calcium ionophore induces on osmiophilic inclusion bodies of 3LL cells similar process of lamellar bodies formation to those reported in the development of the osmiophilic inclusions bodies within the granular pneumocyte of normal lungs. HPLC analysis shows that calcium ionophore generates quantitative changes in the 3LL cytoplasmic protein content expressed by an increase of 18-22 kDa and 4-9 kDa proteins.

Topics: Animals; Calcimycin; Cytoplasm; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Proteins; Tumor Cells, Cultured

We studied the generation and metabolism of leukotrienes (LTs) in human lung macrophages obtained from lung tissue of patients with central bronchial carcinoma. By counterflow centrifugation macrophages were enriched with a purity of more than 95-100%. A time and dose dependent generation of LTB4 and LTC4 was determined by specific radioimmunoassays after stimulation with the Ca-ionophore and anti-IgE. The amount of LTB4 exceeded the amount of LTC4. The concentrations of leukotrienes in the macrophage fraction amounted to 4.3 +/- 2.2 ng LTB4 and 0.6 +/- 0.05 ng LTC4/1 x 10(7) cells after 5 min of incubation with the Ca-ionophore. The LTB4 levels decreased to 3.0 +/- 0.6 ng after 60 min indicating the metabolism of the generated LTB4 by human lung macrophages. This was confirmed by incubation of the cells with exogenously added [3H]LTB4. LTB4 was converted into unpolar products as was identified by thin-layer chromatography and high-performance liquid chromatography; a comparison with the fibroblast cell line L929 which is known to convert LTB4 into the dihydro-LTB4 metabolite (5,12-dihydroxyeicosatrienoic acid) indicates that human lung macrophages use the same pathway of metabolization. Biological inactivation as determined by chemotaxis and cross-reaction with the LTB4 antiserum correlates with the degree of LTB4 metabolism. Moreover, the macrophages convert LTC4 into LTD4 and LTE4 by the enzymatic activity of the gamma-glutamyltranspeptidase and dipeptidase. Our data emphasize that the human alveolar macrophage not only produces arachidonic acid metabolites but modulates the local inflammatory potential by its metabolizing capacity for leukotrienes.

Topics: Antibodies, Anti-Idiotypic; Calcimycin; Carcinoma, Bronchogenic; Cells, Cultured; Humans; Immunoglobulin E; Leukotriene B4; Lung; Lung Neoplasms; Macrophages; SRS-A

Activation of cell phospholipase, release of arachidonic acid and stimulation of prostaglandin synthesis were studied in a newly described human tumor cell line (Lu-65). In the Lu-65 tumor cell line, the calcium ionophore A23187 (2 microM) caused a 100% increase in the release of 3H-arachidonic acid and a 7-fold increase in the synthesis of prostaglandin E2. 1-oleoyl, -2-acetyl-glycerol (100 microM) increased arachidonate release and prostaglandin E2 synthesis by 100%. A23187 and the protein kinase C activators, 1,2-dioctanoyl-glycerol and 1-oleoyl, -2-acetyl-glycerol, decreased the specific radioactivity of 3H-arachidonate in phosphatidylinositol by 37% and 57%, respectively. The effects of A23187 were blocked in Ca2+-free media or in the presence of the phospholipase A2 inhibitor, p-bromophenacyl bromide, while those of 1-oleoyl, -2-acetyl-glycerol were not. The data provide evidence in a human tumor cell line for calcium/phospholipase A2-dependent and independent pathways for arachidonic acid release, both of which preferentially hydrolyze phosphatidylinositol.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Diglycerides; Dinoprostone; Enzyme Activation; Humans; Lung Neoplasms; Phosphatidylinositols; Phospholipases A; Phospholipases A2; Protein Kinase C; Tumor Cells, Cultured

The metabolism of arachidonic acid (AA) was studied in two pulmonary bronchioloalveolar-carcinoma cell lines (NCI-H322 and NCI-H358) and two small cell lung carcinoma cell lines (NCI-H69 and NCI-H128). Exogenous AA was metabolized only in the NCI-H322 and NCI-H358 cells. There was no detectable metabolism of AA in NCI-H69 or NCI-H128 cells, either in the presence or the absence of the calcium ionophore A23187. The major metabolite of AA isolated from both NCI-H322 and NCI-H358 cells was prostaglandin E2 (PGE2). Prostaglandin endoperoxide synthase activities, expressed as immunoreactive PGE2 (pmol/min/mg protein), were 10.3 +/- 0.28 (SD) and 4.8 +/- 0.48 in NCI-H358 and NCI-H322 cells, respectively. The rate of production of PGE2 by both NCI-H358 and NCI-H322 cells was linear up to 10 min. Production of PGE2 in both cell lines was dependent upon substrate concentration and was maximal above 17 microM AA. Moreover, PGE2 did not undergo further metabolism by either the NCI-H358 or the NCI-H322 cells. Aspirin (0.1 mM), a cyclooxygenase inhibitor, decreased PGE2 production by 77 and 60% in NCI-H358 and NCI-H322 cells, respectively. In the presence of exogenous AA the calcium ionophore, A23187 (20 microM), stimulated PGE2 production in NCI-H322 cells by almost 2-fold, although it did not affect PGE2 production in the NCI-H358 cells. In contrast, A23187 stimulated the endogenous production of PGE2 in both NCI-H322 and NCI-H358 cells by 4- and 9-fold respectively. In addition, both the NCI-H358 and NCI-H322 cell lines were susceptible to the cytotoxic effects of the anticancer agent mitoxantrone in both a time and concentration dependent manner. In contrast, the two cell lines lacking detectable prostaglandin synthesis activity, NCI-H69 and NCI-H128 were unaffected by treatment with mitoxantrone. These results illustrate that there are major differences in the abilities of human lung cancer cell lines to biosynthesize and release PGE2. It is conceivable that such differences might have exploitable diagnostic and/or therapeutic implications.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Arachidonic Acid; Arachidonic Acids; Aspirin; Calcimycin; Carcinoma, Small Cell; Cell Line; Dinoprostone; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Mitoxantrone; Prostaglandins E; Time Factors

Topics: Biotransformation; Calcimycin; Cysteine; Glutathione; Glutathione Disulfide; Humans; Lung; Lung Neoplasms; SRS-A

The A549 cell line is a continuous cell line derived from a human adenocarcinoma of the lung. At low cell population density the cells contain relatively few lamellar bodies, but in mature cells in very confluent cultures lamellar bodies are abundant. The lamellar bodies from these cells are enriched for phosphatidylcholine and disaturated phosphatidylcholine. In mature cells, 45% of newly synthesized phosphatidylcholine is disaturated. Stimulation with the calcium ionophore A23187 produces exocytosis of phosphatidylcholine (46% disaturated). The A549 cell synthesizes, stores in lamellar bodies, and secretes phosphatidylcholine, and thus has many important biological properties of the alveolar epithelial type II cell.

Topics: Adenocarcinoma; Calcimycin; Cell Line; Humans; Lung Neoplasms; Phospholipids; Pulmonary Alveoli